Fluoride activation of the Rho family GTP-binding protein Cdc42Hs.
Aluminum tetrafluoride (AlF4-) activation of heterotrimeric G-protein alpha-subunits is a well established aspect of the biochemistry of these proteins; however, until recently it has been thought that AlF4- does not mediate effects on the Ras superfamily of low molecular weight GTP-binding proteins. Recent work demonstrating aluminum fluoride-induced complex formation between Ras and its GTPase-activating proteins (RasGAP and NF1) has provided important insights into the mechanism of GAP-stimulated GTP hydrolysis. We have characterized the AlF4--induced complex formation between the GDP-bound form of the Rho subfamily G-protein Cdc42Hs and a limit functional domain of the Cdc42-GAP using a variety of biochemical techniques. Our results indicate that the apparent affinity of GAP for the AlF4--mediated complex is similar to the affinity observed for the activated (GTP-bound) form of Cdc42 and that beryllium (Be) can replace aluminum in mediating fluoride-induced complex formation. Additionally, the AlF4--induced interaction is weakened significantly by the catalytically compromised GAP(R305A) mutant, indicating that this arginine is critical in transition state stabilization. Unlike Ras, we find that AlF4- and BeF3- mediate complex formation between Cdc42Hs.GDP and downstream target/effector molecules, indicating that there are important differences in the mechanism of effector binding between the Ras and Rho subfamily G-proteins.
Hoffman
G R
GR
Department of Pharmacology, Cornell University, Ithaca, New York 14853, USA.
Nassar
N
N
Oswald
R E
RE
Cerione
R A
RA
D017607:Aluminum Compounds / Q000494:pharmacology*; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002850:Chromatography, Gel; D005454:Fluorescence Polarization; D005459:Fluorides / Q000494:pharmacology*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D020690:GTPase-Activating Proteins; D006160:Guanosine Triphosphate / Q000378:metabolism; D006868:Hydrolysis; D009682:Magnetic Resonance Spectroscopy; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism*; D020764:cdc42 GTP-Binding Protein; D020703:ras GTPase-Activating Proteins
D017607:Aluminum Compounds; D018797:Cell Cycle Proteins; D020690:GTPase-Activating Proteins; D011506:Proteins; D020703:ras GTPase-Activating Proteins; C050992:tetrafluoroaluminate; D006160:Guanosine Triphosphate; D019204:GTP-Binding Proteins; D020764:cdc42 GTP-Binding Protein; D005459:Fluorides